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We narrowed to 939 results for: TIM

Showing: 901 - 920 of 939 results

  1. Western Blot

    Type
    Protocol
    ...buffer and optimal antibody concentrations. Consider titrating your antibody to determine the optimal dose....approaches. Last Update: January 24, 2022 Workflow Timeline Day 1: Prepare lysates, run SDS-PAGE, transfer... concentration in blocking buffer. Pro-Tip The optimal concentration will vary between antibodies but ...detect the bands. Tips and Troubleshooting The optimal lysis buffer will vary depending on the sample ... buffers to find the best for your target. The optimal concentration and blocking buffer will vary between...consider titrating your antibody to determine the optimal dose. To ensure that your antibody is both functioning...

  2. Transfection for Recombinant Antibodies

    Type
    Protocol
    ...regulations. Last Update: February 18, 2022 Workflow Timeline Day 1: Seed cells Day 2: Transfect cells Day 3...no more than 200 µL of NaOH or 20 µL of HCl at a time. Add deionized water to a final volume of 1 L and...resuspended before sampling. Gently swirl the flask 5–10 times before sampling. Transfer 10 µL of trypan blue into...microfuge tube containing the trypan blue. Pipette 10 times to mix. Load 10 µL of the cell suspension/trypan...the tube and vortex for 5 s to mix. Pro-Tip The optimal ratio of DNA:PEI may vary significantly and should...the flask dropwise. Cap the flasks and swirl 5–10 times to mix. Return the flask to the incubator. Section... Feed. Pro-Tip The feed can be repeated up to 4 times for a total of 16% of the culture volume. At 72-...

  3. General Transfection

    Type
    Protocol
    ...reagents. Workflow Timeline Day 0: Seed HEK293T cells (or a subclone of HEK293T optimized for viral production...this protocol with a subclone of HEK293T cells optimized for viral production (AAVpro or Lenti-X), but ...addition of acid or base. Add only a few drops at a time, allow them to mix and recheck the pH to prevent...levels of virus. HEK293T cells should be split 3 times a week: Monday: Plate 1x10 6 cells in a 75 cm 2 ...are below passage 20 for viral production. The optimal mass DNA:mass PEI ratio will need to be empirically...

  4. Protocol - Bacterial Transformation

    Type
    Protocol
    ... cells, which come frozen and are prepared for optimal transformation efficiencies upon thawing. For the...flicking the bottom of the tube with your finger a few times. Pro-Tip Transformation efficiencies will be approximately... Pro-Tip This outgrowth step allows the bacteria time to generate the antibiotic resistance proteins encoded...plates at 37°C overnight. Tips and FAQ How can I save time when carrying out transformations? If you are not...grow up more of the plasmid) you can save a lot of time by shortening or skipping many steps and will still...your transformation procedure is working. TIP: Sometimes less is more. Although it may be counter-intuitive...

  5. Protocol - How to Ligate Plasmid DNA

    Type
    Protocol
    ...ratios to optimize the ligation reaction. See Tips and FAQ below for details on optimization. Incubate...complementary, the two pieces of DNA connect and ultimately are fused by the ligation reaction. The example...is a good idea to take a fresh tube, thaw it one time and aliquot individual tubes of 5, 10 or 20μL for...plasmid in ligation or transformation reagents Optimizing the Vector:Insert Ratio Although a 3:1 insert...to vector ratio is usually sufficient, you can optimize the amount of insert and vector to improve ligation...

  6. Virus Protocol - Generating Stable Cell Lines

    Type
    Protocol
    ...be adapted to alternative cell lines. Workflow Timeline Day 0: Seed and transduce Cells Day 2–3 (am): ...cycles. Procedure Before beginning, determine the optimal dose of selective reagent for your target cell ...that they can grow out in a reasonable amount of time, but not so many that they vastly outnumber the ...important to do regular media changes and maintain optimal growth conditions for the surviving cells. Even... selection while the population expands. At this time, some researchers reduce the concentration of the...has the most desirable level of expression. Over time, transgene expression in a polyclonal population...

  7. Protocol - How to Create a Bacterial Glycerol Stock

    Type
    Protocol
    ...However, if you want to store bacteria for a longer time, you will need to establish glycerol stocks. The...plasmid DNA prep the following day. Tips and FAQ The optimal concentration of long-term glycerol storage is ...glycerol. You can prepare the glycerol stock the same time you prepare your plasmid DNA. In the morning, when...not to freeze/thaw your glycerol stock too many times. Placing the glycerol stock on dry ice while streaking...that you shake the glycerol before freezing (5-6 times). Make sure that you see one uniform solution, and...

  8. Lentivirus ddPCR Titration

    Type
    Protocol
    ...regulations. Last Update: July 7, 2023 Workflow Timeline Day 1: Seed and transduce cells Day 4: Treat cells...When adding the virus to the diluent, pipette 10 times, to remove virus from pipette tip. To mix the dilution...set the P200 pipette to 200 µL and pipette 10–20 times. Use a new pipette tip to transfer 200 µL of the...seeding. Mix each well with a 1 mL pipette 5–10 times. The final volume in the well is 1.5 mL, and the...appropriate PCR tubes. Pipette back and forth 10 times to mix. Generating the Droplets Place a DG8 cartridge...following PCR parameters: Cycling Step Temperature (°C) Time (min) Ramp Rate (°C/sec) # Cycles Enzyme Activation...

  9. Lentivirus Production

    Type
    Protocol
    ... such as stable-cell line generation. Workflow Timeline Day 0: Seed 293T packaging cells Day 1 (pm): Transfect...addition of acid or base. Add only a few drops at a time, allow them to mix and recheck the pH to prevent...discard the tube and thaw a new working stock. The optimal mass DNA:mass PEI ratio will need to be empirically... high viral titer. 293T cells should be split 3 times a week: Monday: Plate 1×10 6 cells in a T75 flask... *Plasmid concentrations and ratios should be optimized for each transfer plasmid. Pro-Tip Endotoxins ...

  10. Affinity Purification of Recombinant Antibodies with Protein A or Protein G

    Type
    Protocol
    ...affinity purify recombinant antibodies. Workflow Timeline Day 1: Purify antibody Day 2 or later: Buffer ... discarding buffer from the collection tube each time. Place column in a new 50 mL LoBind collection tube...space in the column with PBS and pipette several times to mix. Centrifuge at 3100 x g for 8 min . Discard...container. Repeat steps 29-31 at least 4 additional times to ensure a full buffer exchange. Discard the flow...space in the column with PBS and pipette several times to mix. Centrifuge at 3100 x g for 8 min . Remove...

  11. AAV ddPCR Titration

    Type
    Protocol
    ... in the 48-well dilution plate and pipette 5–10 times to mix. Dilute the virus as follows: Mix dilutions...generating aerosols. Be sure to change tips each time. Dilution 1 (20X): 5 µL in 95 µL 1X PCR buffer (...and mix by pipetting the liquid up and down 10–20 times. Gently cover the entire dilution plate with Microseal...appropriate PCR tubes. Pipette back and forth 5 times. Lightly cap the PCR tubes. Generate the Droplets...following PCR parameters. Cycling Step Temperature (°C) Time (min) Ramp Rate (°C/sec) # Cycles Denaturation 95...

  12. Protocol - How to Inoculate a Bacterial Culture

    Type
    Protocol
    ...If so, you will likely need to grow for a longer time to get the correct density of bacteria since they...cell) and they need to grow for longer periods of time (approximately 18–30 h). On the other hand, smaller...What went wrong? Try growing the culture for more time. Some bacterial cultures grow more slowly. Also,...rather than 37 °C often require longer incubation times. Double check that the antibiotic in your LB media...

  13. Molecular Biology Protocol - Restriction Digest of Plasmid DNA

    Type
    Protocol
    ...double digest (digesting with two enzymes at the same time), you will need to determine the best buffer that...and the amount of DNA in the reaction, incubation time can range from 45 mins to overnight. For diagnostic... of methylation sensitive restriction sites . Sometimes enzymes cut sequences which are similar, but not...amount of Master Mix to each tube. This will save you time and ensure consistency across the reactions....

  14. Colony Formation Titering Assay

    Type
    Protocol
    ...the same time as the virus-mediated transduction. For some cell lines, transduction is optimized if the ...cannot be used for later experiments. Workflow Timeline Day 0: Seed and transduce cells Day 2: Replace...that case, polybrene should only be added at the time of viral transduction, and not during the cell seeding...

  15. AAV Purification by Iodixanol Gradient Ultracentrifugation

    Type
    Protocol
    ...column gradient for AAV purification. Workflow Timeline Day 1: Purify Day 2: Buffer exchange and concentration... a T70i rotor at 10 °C. Pro-Tip If you need more time, you can alternatively centrifuge for 2 h at 200,000...Repeat for each QuickSeal tube. (Optional) For first time users, it is a good idea to assay each fraction ...sample and make sure to pipet back and forth a few times to mix the iodixanol that has settled at the bottom...

  16. Protocol - Over-Agar Antibiotic Plating

    Type
    Protocol
    ...through the use of a selection curve to determine optimal antibiotic concentration. Last Update: Oct. 27,...30 minutes with the lid on to give the antibiotic time to more fully absorb. During the incubation, transform...Shown below are the results from an experiment optimizing the concentration of carbenicillin, plated over-agar...

  17. Water Bath Protocol

    Type
    Protocol
    ...liter. Place a thermometer in the water bath. Oftentimes, the water bath will have a spot to secure the...that it will reach the desired temperature by the time you need it for your experiment. Water baths tend... the water bath, place the lid back on. Note the time that you’ve put your materials in the water bath...

  18. Lab Safety for Biosafety Levels One and Two

    Type
    Protocol
    ...protective equipment (PPE) and wear it the whole time you are working in the lab. Do not eat, drink, chew...supervisor what is available and the appropriate contact times for each agent. Make sure you have enough space ...laboratory. Don’t mouth pipette! There may be some times when you may be working with a protocol that requires...

  19. Using a Light Microscope Protocol

    Type
    Protocol
    ...magnifies the image four times the actual size, the 10x objective magnifies it 10 times, and so on. The ocular...focus, use the stage arm to move the stage (and ultimately your slide) until your field of view is centered...

  20. Protocol - How to Run an Agarose Gel

    Type
    Protocol
    ...approximately 75-80% of the way down the gel. A typical run time is about 1-1.5 hours, depending on the gel concentration... use long-wavelength UV and expose for as little time as possible to minimize damage to the DNA. Note:...the gel at a lower voltage for a longer period of time; b) using a wider/thinner gel comb; or c) loading...
Showing: 901 - 920 of 939 results